ABSTRACT The antimicrobial effects of selected medicinal plants commonly used by herbal practitioners in central province Kenya was evaluated on different bacterial strains-Staphylococcus aureus (Gram +ve cocci)-ATCC 20591， Salmonella typhi (Gram –ve rod)-ATCC 2202， Escherichia coli (Gram-ve rod)-STD. 25922， Klebsiella pneumoniae (clinical isolate) and Pseudomonas aeroginosa (Gram-ve rod)-ATCC 25852. Also Candida albicans ATCC EK138 was used as a fungal isolate. Methanol was used as the only solvent in the extraction. The in vitro antimicrobial activity was performed by agar disc diffusion method. The most susceptible Gram-positive bacteria was S. aureus (between 19.33-23.33mm)， while the most susceptible Gram-negative bacteria was P. aeruginosa (14.66-19.33mm). All the extracts showed sufficient inhibitory activity to the test strains. The Gram positive strain (S. aureus) was more sensitive to the extracts (range 23.33-19.33mm) than the Gram negative strains (range 21.00-14.66mm). The mean inhibition value was between 15.997mm and 19.995mm. Statistical analysis revealed that Hyptis spicigera and Crotalaria quartiniana produced significantly different (P≤0.05) zones of inhibition in all the test strains. Other extracts average zones of inhibition showed no significant difference among the test strains. The significant antibacterial activity of active extracts was compared with the standard antimicrobials (Fluconazole for C. albicans， and amoxicillin for bacterial isolates) and dried methanol discs， giving a pooled SD of 2.349mm. The results obtained in the present study suggest that the extracts can be used in treating diseases caused by the test organisms.

In Africa more than 70% of the people refer to ethnomedicine for their health issues. With the emergence of new diseases and drug resistance to infections， traditional medicine should be given more attention in modern research and development. Tuberculosis (TB)， a deadly infectious disease that annually kills about 3 million people worldwide is complicated. This is due to significant toxicity， emergence of multidrug resistant TB (MDR-TB) and extensively drug resistant TB (XDR- TB) and lengthy therapy which creates poor patient compliance. There is also a major therapeutic problem due to emergence of Escherichia coli， Klebsiella pneumoniae and other Blactamase producers. Diarrhoeal diseases are responsible for 4.6 million deaths every year. These highlight the need to develop novel drugs. Natural products provide unlimited opportunities for new drug leads because of the unmatched chemical diversity. This study evaluated the antimicrobial potential of 34 medicinal plants used by communities living around the Lake Victoria region and the Samburu Community of northern Kenya， following an ethnobotanical survey. Plants were collected and identified at the Department of Pharmacy and Complimentary Alternative Medicine， Kenyatta University， Nairobi， Kenya; in whose herbarium voucher specimens were deposited. Methanolic extracts from plants were tested against four strains of Mycobacteria (Mycobacteria tuberculosis， M. kansasii， M. fortuitum， and M. smegmatis) obtained from Kenya Medical Research Institute (KEMRI)， Nairobi， Kenya. BACTEC MGIT 960 system was used. Salmonella typhi (clinical isolate)， Klebsiella pneumoniae (clinical isolate)， Pseudomonas aeruginosa (ATCC 25852)， Escherichia coli (ATCC 25922) Staphylococcus aureus (ATCC 20591) and Candida albicans (ATCC EK138)， obtained from Kenyatta National Hospital in Nairobi， Kenya， were also screened against using standard procedures. The crude extracts were analyzed for presence of phytochemicals. Croton macrostachyus， Vernonia amygdalina， Toddalia asiatica， Aloe secundiflora， Cordia sinensis， and Euphorbia scarlatina gave strong antimycobacterial activity (zero GUs) against M. kansasii， at all concentrations used. Entada abysinnica， T. asiatica， Salvadora persica， C. sinensis， Scadoxus multiflorus and E. scarlatina extracts were active (zero GUs) against M. tuberculosis. Extracts from Carissa edulis， V. amygdalina， A. secundiora， Pistacia aethiopica， S persica， S. multiflorus， E. scarlatina， and Acacia nilotica were active (zero GUs) against M. fortuitum. Against M. smegmatis， Carissa edulis， V. amygdalina， A. secundiora， S. persica， S. multiflorus， E. scarlatina and A. nilotica were active (zero GUs). Eurphobia scarlatina was active (Zero GUs) against all the strains of mycobacteria. There was significant difference of the means of the zones of inhibition of the S. typhi， K. pneumoniae， P. aeruginosa， E. coli， S aureus and C. albicans at P< 0.05. The MICs and the MBCs of the extracts were determined by use of microtitre plate method with E. abysinnica， T. asiatica， Thylachium africanum， A. secundflora， A. nilotica and Momordica charantia extracts showing good activity with MICS and MBCS of 4.687- 18.75 mg/ml in some test cultures. Klebsiella pneumoniae and C. albicans were mostly insensitive to extracts. Preliminary phytochemistry identified six phytochemicals to which tannins were common in most plant extracts. The data suggests that plant extracts could be a rich source of antimicrobial agents. Results also provide an indication of merit in their ethnomedicinal use

Feature Article Free Effects of Pulsed Electromagnetic Field Frequencies on the Osteogenic Differentiation of Human Mesenchymal Stem Cells Fei Luo， PhD; Tianyong Hou， PhD; Zehua Zhang， PhD; Zhao Xie， PhD; Xuehui Wu， PhD; Jianzhong Xu， PhD Orthopedics April 2012 - Volume 35 · Issue 4： e526-e531 Posted April 1， 2012 DOI： 10.3928/01477447-20120327-11 READ/SUBMIT COMMENTS EMAIL PRINT SAVE READ OR SUBMIT ARTICLE COMMENTS EMAIL SAVE if (typeof (savedoc) != "function") { function savedoc() { mustlogin(); } } Abstract Article Figures/Tables References Abstract The purpose of this study was to evaluate the effect of different frequencies of pulsed electromagnetic fields on the osteogenic differentiation of human mesenchymal stem cells. Third-generation human mesenchymal stem cells were irradiated with different frequencies of pulsed electromagnetic fields， including 5， 25， 50， 75， 100， and 150 Hz， with a field intensity of 1.1 mT， for 30 minutes per day for 21 days. Changes in human mesenchymal stem cell morphology were observed using phase contrast microscopy. Alkaline phosphatase activity and osteocalcin expression were also determined to evaluate human mesenchymal stem cell osteogenic differentiation. Different effects were observed on human mesenchymal stem cell osteoblast induction following exposure to different pulsed electromagnetic field frequencies. Levels of human mesenchymal stem cell differentiation increased when the pulsed electromagnetic field frequency was increased from 5 hz to 50 hz， but the effect was weaker when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 hz. The most significant effect on human mesenchymal stem cell differentiation was observed at of 50 hz. The results of the current study show that pulsed electromagnetic field frequency is an important factor with regard to the induction of human mesenchymal stem cell differentiation. Furthermore， a pulsed electromagnetic field frequency of 50 Hz was the most effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro. Drs Luo， Hou， Zhang， Xie， Wu， and Xu are from the Department of Orthopaedics， South-West Hospital， The Third Military University， Chongqing， China. Drs Luo， Hou， Zhang， Xie， Wu， and Xu have no relevant financial relationships to disclose. This study was supported by the National High Technology Research and Development Program (863 Project， Grant No.： 2006AA02A122) and the Chongqing Natural Sciences Foundation (Grant No.： 2010JJ0379). Correspondence should be addressed to： Jianzhong Xu， PhD， Department of Orthopaedics， South-West Hospital， The Third Military University， No. 29 Gaotanyan Rd， Chongqing 400038， China (xjzslw@hotmail.com). Bone tissue engineering involves the use of tissue engineering methods to promote the generation and differentiation of bony cells. Using this approach， new approaches can be explored to repair long segmental bone defects. Mesenchymal stem cells are one of the most widely used types of stem cells for bone tissue engineering. Following in vitro amplification using chemical induction to induce mesenchymal stem cell osteoblast differentiation， the resulting osteoblasts and support material can be combined to generate engineered bone tissue. In 1977， Bassett et al 1 developed pulsed electromagnetic field therapy and successfully treated a group of patients with bone nonunion. Since then， several studies have reported that pulsed electromagnetic fields accelerate the speed of mesenchymal stem cell amplification and osteoblast induction， growth factor secretion， and extracellular matrix synthesis; pulsed electromagnetic fields may also promote bone reconstruction and accelerate bone growth. 2–7 These findings suggest that further studies of the effects of pulsed electromagnetic fields on mesenchymal stem cell osteoblast differentiation and bone tissue engineering are needed. 8–13 These previous studies were performed using a specific pulsed electromagnetic field frequency during the course of the experiments; notably， the frequency is an important indicator for determining the biological effects of pulsed electromagnetic fields. In the current study， we investigated the effects of different pulsed electromagnetic field frequencies on mesenchymal stem cell induction in vitro with the overall goal of providing a new approach for mesenchymal stem cell induction in vitro and experimental support for a new type of bioreactor and the clinical application of pulsed electromagnetic fields to promote bone fracture healing. The results of the current study should provide a strong theoretical foundation for future pulsed electromagnetic field-related research. Materials and Methods The following solutions， kits， and equipment were used in the experiments described herein： Percoll solution (Sigma-Aldrich， St Louis， Missouri)， HyClone Dulbecco’s Modified Eagle Medium： Nutrient Mixture F-12 (DMEM/F12) (1：1) (Thermo Fisher Scientific Inc， Waltham， Massachusetts)， HyClone fetal calf serum (Thermo Fisher Scientific Inc)， alkaline phosphatase test kit (Nanjing Jiancheng Bioengineering Institute， Nanjing， China)， osteocalcin radioimmunity kit (Beijing China Atomic Research Institute， Beijing， China)， mouse-anti-human osteocalcin antibody (Boshide， Wuhan， China)， ultraviolet/visible spectrophotometer (UV751GD; the Shanghai Equipment Confactory， Shanghai， China)， and controllable pulsed electromagnetic field activator (Logistical Engineering University of PLA， Chongqing， China). Separation and Cultivation of Human Mesenchymal Stem Cells Bony marrow (5–10 mL) from both sides of the iliac crest was collected from healthy volunteers in a sterilized environment using heparin as the anticoagulant. Percoll solution (1.073 g/mL) was used to isolate the bony marrow. A total of 5×105 cells/cm 2 were inoculated into a culture flask containing DMEM/F12 supplemented with 15% fetal calf serum， and the cells were incubated under saturated humidity. After 3 generations， the cells were digested and centrifuged to generate a cell suspension， which was divided into 6 experimental groups and 1 control group. Treatment of Human Mesenchymal Stem Cells With Different Pulsed Electromagnetic Field Frequencies The 6 experimental cell groups were treated with a Helmholtz coil with pulsed electromagnetic fields， which was a dual coil with a 10-cm space between the 2 coils. The Hall effect was used to measure the magnetic reactor to confirm that the field’s homogeneity and stabilization were acceptable. The coil was placed into the cell incubator， and the field was set to different pulsed electromagnetic field frequencies， including 5， 25， 50， 75， 100， and 150 Hz， each with a field intensity of 1.1 mT， for 30 minutes per day for 21 days. The control groups of cells were incubated under the same experimental conditions with no exposure to the pulsed electromagnetic fields. Morphological Observations Changes in cell morphology under the different growth conditions were observed using an inverted microscope. Photos of cell morphology were taken on days 1， 3， 5， and 7 after serial subcultivation. Ultramicrostructural Observations Following exposure to pulsed electromagnetic fields， when the cells covered 80% of the area of the culture bottle， they were digested， centrifuged， and washed twice with phosphate buffered saline. The cells were immobilized with 2.5% glutaraldehyde and subjected to gradient acetone anhydration， followed by embedding in epoxy resin. Extra thin sections were generated and stained with uranyl acetate and lead citrate. Transmission electron microscopy was used to observe the ultramicrostructure of the experimental and control groups. Alkaline Phosphoric Acid Enzyme Staining When using Gomori stain， cells positive for alkaline phosphatase expression are brownish-black. Following 6 days of pulsed electromagnetic field stimulation， the human mesenchymal stem cells were removed from the incubator， rinsed with phosphate buffered saline， fixed in ice-cold acetone (−20°C)， incubated for 3 hours at 37°C in phosphoric acid enzyme liquids， washed with distilled water， and subjected to ammonium sulfide processing using 2% nitric acid and 1% cobalt， 1 by 1 in order， followed by staining with neutral and red contrast dye. Alkaline Phosphatase Activity Measurements After 3， 6， 9， 12， and 15 days of pulsed electromagnetic field exposure， cells grown in 12-well flat-bottomed plates from each group were digested， centrifuged， collected， and washed twice with phosphate buffered saline. The cell density was adjusted to 1×105 cells/mL in 0.5% Triton-X100 (Sigma-Aldrich， St Louis， Missouri)， and the cells were incubated at 4°C for 12 hours. Intermittent ultrasound exposure on ice was used to break up the cells and ensure complete cell lysis (150 W， 5 s). The absorbance of 50 μL of the cell lysate was examined at 520 nm to determine the A value， and a standard formula was used to calculate alkaline phosphatase activity. Collagen Type I and Osteocalcin Immunostaining Coverslips were removed after 12 days， followed by phosphate buffered saline rinses， fixation with 95% ethanol， and treatment with 3% hydrogen dioxide， followed by rinses with distilled water and incubation with 5% normal goat serum. Cells were subjected to immunohistochemical staining using the streptavidin-biotin peroxidase complex method with a rat anti-collagen type I and anti-osteocalcin monoclonal antibody. Quantification of Osteocalcin Levels At 7， 14， and 21 days， 50 mL were removed from each culture， followed by digestion with diastase vera. Following centrifugation， a total of 1×106 cells were resuspended in 1 mL of a 1：1 admixture of phosphate buffered saline and Triton-X100， followed by incubation at 4°C overnight. The resulting samples were sent to the Atomic Medical Department of Southwest Hospital to measure the osteocalcin content in the cells using a radiographic-immunity method based on the radiographic-immunity competitive binding principle. In this assay， 125 I-marked osteocalcin competes with free osteocalcin for binding to the osteocalcin antibody， and a ϒ counter is used to measure the sediment counts per minute， which is used to determine the osteocalcin content of the sample according to a standard curve. Staining With Alizarin Monosulfonate Calcium Dye After the appearance of oval-shaped nodules in the 6-well culture plate， the coverslips were removed， rinsed with phosphate buffered saline， fixed with 75% alcohol for 30 minutes， stained with 2% alizarin monosulfonate， dehydrated using an alcohol gradient， made transparent with xylene， and mounted using neutral resin. Staining With Von Kossa Calcium Dye After oval-shaped nodules appeared in the 6-well culture plate， the coverslips were removed， rinsed with phosphate buffered saline， fixed with 75% alcohol， immersed in 2% silver nitrate aqua， exposed to ultraviolet light for 30 minutes， washed with distilled water， stained with neutral red dye， treated with an alcohol gradient， made transparent with dimethyl benzene， and sealed using a neutral resin. Staining With Achromycin Dye After oval-shaped nodules appeared in the 6-well culture plate， the coverslips were removed， treated with 0.1 mg/mL achromycin culture solution for 30 minutes， incubated with a common culture solution for 30 minutes， rinsed with phosphate buffered saline， fixed with 75% alcohol， and observed using a fluorescence microscope. Statistical Analyses SPSS version 11.0 software (IBM， Armonk， New York) was used for statistical analyses. Self-paired t analysis was used to analyze each set of experimental data. One-factor analysis of variance was used to analyze different groups stimulated with different pulsed electromagnetic fields， and F analysis was used to complete the statistical analysis. Results Inverted Phase Contrast Microscope Observations At 4 hours post-inoculation， the third-generation human mesenchymal stem cells were adhered to the plates， and they began to divide and grow 24 hours after they adhered. After 3 days， pulsed electromagnetic field group cells were larger than control group cells， and their morphology continued to change; the cells eventually became triangular and polygonal in shape， scales formed， and the cytoplasm contained abundant matrix and granular material. No obvious differences were observed in the appearances of pulsed electromagnetic field group cells compared with control group cells. Over time， the cells became confluent and began to exhibit overlapping growth. After 3 weeks， mineralization of the matrix led to the fusion of the oval-shaped calcified nodules. Around the nodules， the cells were distributed in an array-like pattern. Ultramicrostructural Observations Transmission electron microscopy analysis of human mesenchymal stem cells in the pulsed electromagnetic field group showed that they were more differentiated than the control group cells. The nuclear matrix ratio of pulsed electromagnetic field group cells was lower than that of control group cells. The cytoplasm of pulsed electromagnetic field group cells contained abundant organelles， including mitochondria， rough endoplasmic reticulum， and Golgi bodies. Control group human mesenchymal stem cells were more immature with larger nuclei， a similar nuclear-matrix ratio， and fewer organelles. Alkaline Phosphatase Staining Cells that were not stimulated with pulsed electromagnetic fields were negative for alkaline phosphatase expression， whereas cells subjected to pulsed electromagnetic field stimulation were highly positive for alkaline phosphatase expression， with brownish-black cytoplasm and black granulated precipitates. Alkaline Phosphatase Activity Measurements Pulsed electromagnetic field group cells exhibited stronger alkaline phosphatase activity than control group cells starting on the third day. As time progressed， alkaline phosphatase activity was higher in pulsed electromagnetic field group cells， reaching a peak at 12 days. By day 15， no great change had occurred， and alkaline phosphatase activity remained stable， although it remained significantly higher than that of the control groups (Figure 1 ). Furthermore， different frequencies were associated with different alkaline phosphatase activity levels. Alkaline phosphatase activity in the 50-Hz pulsed electromagnetic field group was higher than that of the other pulsed electromagnetic field groups on days 9， 12， and 15. Figure 1： Alkaline phosphatase (ALP) activity in human mesenchymal stem cells treated with different frequencies of pulsed electromagnetic fields (PEMF). Collagen Type I and Osteocalcin Immunohistochemical Staining After 12 days of pulsed electromagnetic field stimulation， human mesenchymal stem cells subjected to collagen type I immunohistochemical staining exhibited high levels of yellow granulation and were highly positive. In contrast， no yellow granulation was detected in the control group cells. Osteocalcin immunohistochemical staining of human mesenchymal stem cells showed that they exhibited high levels of yellow granulation and were highly positive. In contrast， no yellow granulation was detected in the control group cells. Quantification of Osteocalcin Levels On day 7， cells in all of the different pulsed electromagnetic field groups expressed osteocalcin slightly， whereas the control group cells did not. On day 14， cells in the pulsed electromagnetic field groups expressed significantly higher levels of osteocalcin; although cells in the control group also expressed osteocalcin on day 14， the osteocalcin levels of control cells were significantly lower than the pulsed electromagnetic field groups. Osteocalcin levels varied among the pulsed electromagnetic field groups treated with different frequencies， and osteocalcin levels in the pulsed electromagnetic field group treated with a frequency of 50 Hz were higher than the others on day 21 (Figure 2 ). Figure 2： Osteocalcin content of mesenchymal stem cells treated with different frequencies of pulsed electromagnetic fields (PEMF). Alizarin Monosulfonate Calcium Nodule Staining Following pulsed electromagnetic field stimulation for 21 days， matrix mineralization led to the formation of oval-shaped calcium nodules， which stained orange around the nodule with alizarin monosulfonate calcium， and the cells around the nodule were distributed in an array-like pattern. In contrast， no calcium nodules were present in the control group cells. Von Kossa Calcium Dye Staining On day 21， cells stimulated with pulsed electromagnetic fields began to exhibit oval-shaped calcium nodules. The nodules changed from transparent to opaque， and then turned into a black mass. After staining with a calcium dye， massive black crystal deposits were identified， which were not present in the control group cells. The results of this analysis showed that pulsed electromagnetic field treatment of human mesenchymal stem cells can stimulate bone induction. Achromycin Fluorescent Labeling Cells stimulated by pulsed electromagnetic fields began to exhibit oval-shaped calcium nodules after 3 weeks in culture， and the nodules increased in size and changed from transparent to an opaque-black mass. After achromycin fluorescent labeling， the nodules appeared golden， and the light was equal to the size and shade under the inverted microscope. In contrast， no calcium nodules were present in the control group. Discussion The construction of tissue-engineered bone requires the establishment of seed cells with strong characteristics. Mesenchymal stem cells， which are derived from mesoderm， are adult stem cells that have strong reproductive activity and are multipotent. The differentiation of mesenchymal stem cells into osteoblasts can be induced by cytokines， hormones， physical methods， and by many other factors. Pulsed electromagnetic field is a method used to treat bony delayed union and bony nonunion. Pulsed electromagnetic field treatment is associated with satisfactory effects， and has recently been used as a therapy to treat inborn bone defects， bone necrosis， bone transplantation， and spinal fusion. 1–6 Previous studies have shown that pulsed electromagnetic fields can mediate extracellular matrix synthesis， increase alkaline phosphatase expression by osteoblasts， and promote the secretion of osteocalcin and collagen. 6，14，15 Mesenchymal stem cells are an important ancestor cell in the process of bone growth. Based on these findings， some researchers have proposed that pulsed electromagnetic fields could induce a similar biological effect on human mesenchymal stem cells and promote bone formation. 8–13 This proposal has gained support based on earlier studies performed in our laboratory. However， prior to the current study， whether different pulsed electromagnetic field frequencies exhibit a differential effect on cell differentiation was not known. In the current study， we sought to determine whether a specific pulsed electromagnetic field frequency is optimal for the purpose of tissue-engineered bone construction. The results of this study have important implications with regard to the development of a new type of bioreactor and the clinical application of pulsed electromagnetic fields to promote bone fracture healing. Among the factors that affect electromagnetic fields， frequency plays a major role. Bassett et al 1 reported that different frequencies of pulsed electromagnetic field influence bone fracture healing. Research has shown that pulsed electromagnetic fields in the frequency between 1 and 100 Hz allow the electromagnetic field to exert a biological effect. 16 In the skeletal system， the endogenous frequency ranges from 1 to 5 Hz (walk frequency) or from 10 to 100 Hz (muscle contraction power frequency); Lee and McLeod 17 proposed that the most effective pulsed electromagnetic field frequency should be similar to that associated with normal body action frequency. In the current study， we referred to a report focused on the different frequency of pulsed electromagnetic fields that affect the union of a bone fracture. In the frequency range of 1 to 150 Hz， we chose 5， 25， 50， 75， 100， and 150 Hz to determine whether different effects occurred on the promotion of human mesenchymal stem cell osteoblast differentiation and to identify the optimal pulsed electromagnetic field frequency. Using an inverted phase contrast microscope to analyze cell morphology， we found that pulsed electromagnetic field treatment resulted in larger cells relative to the control groups. Furthermore， the shape of the cells in the pulsed electromagnetic field group included triangular and polygonal cells with scales. The amount of cytosolic granulomaterial and secreted matrix also increased significantly in the pulsed electromagnetic field group cells. Transmission electron microscopy analysis showed that human mesenchymal stem cells stimulated by pulsed electromagnetic field were more mature than control group cells. After 3 weeks in culture， pulsed electromagnetic field group cells exhibited oval and round nodules and were positive for alizarin staining. Alkaline phosphatase is an enzyme that hydrolyzes organic phosphate during the process of bone construction; this process provides the phosphonic acid required for hydroxyapatite ceramic deposition and promotes bone formation. Alkaline phosphatase expression is a marker for the initiation of bone formation and differentiation. Furthermore， osteocalcin is 1 type of noncollagen protein that is secreted by osteoblasts. When calcium is present， osteocalcin combines with hydroxyapatite and stabilize the conformation， which is regarded as the most distinctive mark of osteoblast differentiation. Thus alkaline phosphatase and osteocalcin are 2 important indices reflecting the osteoblast differentiating into osteocyte and the matrix becoming calcified. 18 When we stimulated human mesenchymal stem cells with different pulsed electromagnetic field frequencies in vitro， all of the cells in each frequency group exhibited detectable alkaline phosphatase expression after 3 days， and with increasing time， alkaline phosphatase expression continued to increase. Taken together， this finding provided evidence that bone differentiation was initiated. After 1 week in culture， cells stimulated with pulsed electromagnetic field expressed osteocalcin， which reached peak levels in the third week at the same time that mineralized nodules appeared. In control group cells， alkaline phosphatase activity was always low， and we detected no osteocalcin expression. These findings provide evidence that pulsed electromagnetic field stimulation of human mesenchymal stem cells leads to bone differentiation. In the current study， different pulsed electromagnetic field frequencies were associated with different levels of bone induction. From 5 to 50 Hz， as the frequency increased， the inductive effect on bone differentiation also increased. However， from 50 to 150 Hz， as the frequency increased， the inductive effect on bone differentiation decreased. Taken together， the results of the current study show that pulsed electromagnetic field frequency is an important factor for the induction of human mesenchymal stem cell bone differentiation. Different frequencies of pulsed electromagnetic field have different effects on the induction of bone differentiation， and 50 Hz was an optimal frequency for the in vitro induction of human mesenchymal stem cell bone differentiation. The results of this study may help promote the osteogenic differentiation of seed cells for tissue engineering for bone production and provide new insights and parameters for clinical applications aimed at bone fracture healing. The biological mechanisms responsible for pulsed electromagnetic field-mediated promotion of bone differentiation by human mesenchymal stem cells remain to be determined and will be the focus of future studies. References Bassett CA， Pilla AA， Pawluk RJ. A non-operative salavage of surgically-resistant pseudarthrosis and non-unions by pulsing electromagnetic fields. A preliminary report. Clin Orthop Relat Res . 1977; (124)：128–143. Simmons JW Jr， Mooney V， Thacker I. Pseudarthrosis after lumbar spine fusion： nonoperative salvage with pulsed electromagnetic fields. Am J Orthop (Belle Mead NJ) . 2004; 33(1)：27–30. Shen WW， Zhao JH. Pulsed electromagnetic fields stimulation affects BMD and local factor production of rats with disuse osteoporosis. Bioelectromagnetics . 2010; 31(2)：113–119. Midura RJ， Ibiwoye MO， Powell KA， et al. Pulsed electromagnetic field treatments enhance the healing of fibular osteotomies. J Orthop Res . 2005; 23(5)：1035–1046. doi：10.1016/j.orthres.2005.03.015 [CrossRef] Chang WH， Chen LT， Sun JS， Lin FH. Effect of pulse-burst electromagnetic field stimulation on osteoblast cell activities. Bioelectromagnetics . 2004; 25(6)：457–465. doi：10.1002/bem.20016 [CrossRef] Li JK， Lin JC， Liu HC， Chang WH. Cytokine release from osteoblasts in response to different intensities of pulsed electromagnetic field stimulation. Electromagn Biol Med . 2007; 26(3)：153–165. doi：10.1080/15368370701572837 [CrossRef] Lin HY， Lin YJ. In vitro effects of low frequency electromagnetic fields on osteoblast proliferation and maturation in an inflammatory environment [published online ahead of print March 29， 2011]. Bioelectromagnetics . 2011; 32(7)：552–560. doi：10.1002/bem.20668 [CrossRef] Schwartz Z， Simon BJ， Duran MA， Barabino G， Chaudhri R， Boyan BD. Pulsed electromagnetic fields enhance BMP-2 dependent osteoblastic differentiation of human mesenchymal stem cells. J Orthop Res . 2008; 26(9)：1250–1255. doi：10.1002/jor.20591 [CrossRef] Tsai MT， Li WJ， Tuan RS， Chang WH. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res . 2009; 27(9)：1169–1174. doi：10.1002/jor.20862 [CrossRef] Sun LY， Hsieh DK， Yu TC， et al. Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells. Bioelectromagnetics . 2009; 30(4)：251–260. doi：10.1002/bem.20472 [CrossRef] Sun LY， Hsieh DK， Lin PC， Chiu HT， Chiou TW. Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation. Bioelectromagnetics . 2010; 31(3)：209–219. Tsai MT， Li WJ， Tuan RS， Chang WH. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res . 2009; 27(9)：1169–1174. doi：10.1002/jor.20862 [CrossRef] Griffin M， Iqbal SA， Sebastian A， Colthurst J， Bayat A. Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model [published online ahead of print August 16， 2011]. PLoS One . 2011; 6(8)：e23404. doi：10.1371/journal.pone.0023404 [CrossRef] Diniz P， Shomura K， Soejima K， Ito G. Effects of pulsed electromagnetic field (PEMF) stimulation on bone tissue like formation are dependent on the maturation stages of the osteoblasts. Bioelectromagnetics . 2002; 23(5)：398–405. doi：10.1002/bem.10032 [CrossRef] Li JK， Lin JC， Liu HC， Chang WH. Cytokine release from osteoblasts in response to different intensities of pulsed electromagnetic field stimulation. Electromagn Biol Med . 2007; 26(3)：153–165. doi：10.1080/15368370701572837 [CrossRef] Muehsam DJ， Pilla AA. The sensitivity of cells and tissues to exogenous fields： effects of target system initial state. Bioelectrochem Bioenerg . 1999; 48(1)：35–42. doi：10.1016/S0302-4598(98)00149-4 [CrossRef] Lee JH， McLeod KJ. Morphologic responses of osteoblast-like cells in monolayer culture to ELF electromagnetic fields. Bioelectromagnetics . 2000; 21(2)：129–136. doi：10.1002/(SICI)1521-186X(200002)21：23.0.CO;2-Q [CrossRef] Kim HK， Cho SG， Kim JH， et al. Mevinolin enhances osteogenic genes (ALP， type I collagen and osteocalcin)， CD44， CD47 and CD51 expression during osteogenic differentiation [published online ahead of print January 3， 2009]. Life Sci . 2009; 84(9–10)：290–295. doi：10.1016/j.lfs.2008.12.017 [CrossRef] Figure 1： Alkaline phosphatase (ALP) activity in human mesenchymal stem cells treated with different frequencies of pulsed electromagnetic fields (PEMF). Figure 2： Osteocalcin content of mesenchymal stem cells treated with different frequencies of pulsed electromagnetic fields (PEMF). References Bassett CA， Pilla AA， Pawluk RJ. A non-operative salavage of surgically-resistant pseudarthrosis and non-unions by pulsing electromagnetic fields. A preliminary report. Clin Orthop Relat Res . 1977; (124)：128–143. Simmons JW Jr， Mooney V， Thacker I. Pseudarthrosis after lumbar spine fusion： nonoperative salvage with pulsed electromagnetic fields. Am J Orthop (Belle Mead NJ) . 2004; 33(1)：27–30. Shen WW， Zhao JH. Pulsed electromagnetic fields stimulation affects BMD and local factor production of rats with disuse osteoporosis. Bioelectromagnetics . 2010; 31(2)：113–119. Midura RJ， Ibiwoye MO， Powell KA， et al. Pulsed electromagnetic field treatments enhance the healing of fibular osteotomies. J Orthop Res . 2005; 23(5)：1035–1046. doi：10.1016/j.orthres.2005.03.015 [CrossRef] Chang WH， Chen LT， Sun JS， Lin FH. Effect of pulse-burst electromagnetic field stimulation on osteoblast cell activities. Bioelectromagnetics . 2004; 25(6)：457–465. doi：10.1002/bem.20016 [CrossRef] Li JK， Lin JC， Liu HC， Chang WH. Cytokine release from osteoblasts in response to different intensities of pulsed electromagnetic field stimulation. Electromagn Biol Med . 2007; 26(3)：153–165. doi：10.1080/15368370701572837 [CrossRef] Lin HY， Lin YJ. In vitro effects of low frequency electromagnetic fields on osteoblast proliferation and maturation in an inflammatory environment [published online ahead of print March 29， 2011]. Bioelectromagnetics . 2011; 32(7)：552–560. doi：10.1002/bem.20668 [CrossRef] Schwartz Z， Simon BJ， Duran MA， Barabino G， Chaudhri R， Boyan BD. Pulsed electromagnetic fields enhance BMP-2 dependent osteoblastic differentiation of human mesenchymal stem cells. J Orthop Res . 2008; 26(9)：1250–1255. doi：10.1002/jor.20591 [CrossRef] Tsai MT， Li WJ， Tuan RS， Chang WH. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res . 2009; 27(9)：1169–1174. doi：10.1002/jor.20862 [CrossRef] Sun LY， Hsieh DK， Yu TC， et al. Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells. Bioelectromagnetics . 2009; 30(4)：251–260. doi：10.1002/bem.20472 [CrossRef] Sun LY， Hsieh DK， Lin PC， Chiu HT， Chiou TW. Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation. Bioelectromagnetics . 2010; 31(3)：209–219. Tsai MT， Li WJ， Tuan RS， Chang WH. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res . 2009; 27(9)：1169–1174. doi：10.1002/jor.20862 [CrossRef] Griffin M， Iqbal SA， Sebastian A， Colthurst J， Bayat A. Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model [published online ahead of print August 16， 2011]. PLoS One . 2011; 6(8)：e23404. doi：10.1371/journal.pone.0023404 [CrossRef] Diniz P， Shomura K， Soejima K， Ito G. Effects of pulsed electromagnetic field (PEMF) stimulation on bone tissue like formation are dependent on the maturation stages of the osteoblasts. 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Life Sci . 2009; 84(9–10)：290–295. doi：10.1016/j.lfs.2008.12.017 [CrossRef] Authors Drs Luo， Hou， Zhang， Xie， Wu， and Xu are from the Department of Orthopaedics， South-West Hospital， The Third Military University， Chongqing， China. Drs Luo， Hou， Zhang， Xie， Wu， and Xu have no relevant financial relationships to disclose. This study was supported by the National High Technology Research and Development Program (863 Project， Grant No.： 2006AA02A122) and the Chongqing Natural Sciences Foundation (Grant No.： 2010JJ0379). Correspondence should be addressed to： Jianzhong Xu， PhD， Department of Orthopaedics， South-West Hospital， The Third Military University， No. 29 Gaotanyan Rd， Chongqing 400038， China ().xjzslw@hotmail.com 10.3928/01477447-20120327-11 pagination Previous Article Next Article Abstract The purpose of this study was to evaluate the effect of different frequencies of pulsed electromagnetic fields on the osteogenic differentiation of human mesenchymal stem cells. Third-generation human mesenchymal stem cells were irradiated with different frequencies of pulsed electromagnetic fields， including 5， 25， 50， 75， 100， and 150 Hz， with a field intensity of 1.1 mT， for 30 minutes per day for 21 days. Changes in human mesenchymal stem cell morphology were observed using phase contrast microscopy. Alkaline phosphatase activity and osteocalcin expression were also determined to evaluate human mesenchymal stem cell osteogenic differentiation. Different effects were observed on human mesenchymal stem cell osteoblast induction following exposure to different pulsed electromagnetic field frequencies. Levels of human mesenchymal stem cell differentiation increased when the pulsed electromagnetic field frequency was increased from 5 hz to 50 hz， but the effect was weaker when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 hz. The most significant effect on human mesenchymal stem cell differentiation was observed at of 50 hz. The results of the current study show that pulsed electromagnetic field frequency is an important factor with regard to the induction of human mesenchymal stem cell differentiation. Furthermore， a pulsed electromagnetic field frequency of 50 Hz was the most effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro. Drs Luo， Hou， Zhang， Xie， Wu， and Xu are from the Department of Orthopaedics， South-West Hospital， The Third Military University， Chongqing， China. Drs Luo， Hou， Zhang， Xie， Wu， and Xu have no relevant financial relationships to disclose. This study was supported by the National High Technology Research and Development Program (863 Project， Grant No.： 2006AA02A122) and the Chongqing Natural Sciences Foundation (Grant No.： 2010JJ0379). Correspondence should be addressed to： Jianzhong Xu， PhD， Department of Orthopaedics， South-West Hospital， The Third Military University， No. 29 Gaotanyan Rd， Chongqing 400038， China ().xjzslw@hotmail.com Article The purpose of this study was to evaluate the effect of different frequencies of pulsed electromagnetic fields on the osteogenic differentiation of human mesenchymal stem cells. Third-generation human mesenchymal stem cells were irradiated with different frequencies of pulsed electromagnetic fields， including 5， 25， 50， 75， 100， and 150 Hz， with a field intensity of 1.1 mT， for 30 minutes per day for 21 days. Changes in human mesenchymal stem cell morphology were observed using phase contrast microscopy. Alkaline phosphatase activity and osteocalcin expression were also determined to evaluate human mesenchymal stem cell osteogenic differentiation. Different effects were observed on human mesenchymal stem cell osteoblast induction following exposure to different pulsed electromagnetic field frequencies. Levels of human mesenchymal stem cell differentiation increased when the pulsed electromagnetic field frequency was increased from 5 hz to 50 hz， but the effect was weaker when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 hz. The most significant effect on human mesenchymal stem cell differentiation was observed at of 50 hz. The results of the current study show that pulsed electromagnetic field frequency is an important factor with regard to the induction of human mesenchymal stem cell differentiation. Furthermore， a pulsed electromagnetic field frequency of 50 Hz was the most effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro. Drs Luo， Hou， Zhang， Xie， Wu， and Xu are from the Department of Orthopaedics， South-West Hospital， The Third Military University， Chongqing， China. Drs Luo， Hou， Zhang， Xie， Wu， and Xu have no relevant financial relationships to disclose. This study was supported by the National High Technology Research and Development Program (863 Project， Grant No.： 2006AA02A122) and the Chongqing Natural Sciences Foundation (Grant No.： 2010JJ0379). Correspondence should be addressed to： Jianzhong Xu， PhD， Department of Orthopaedics， South-West Hospital， The Third Military University， No. 29 Gaotanyan Rd， Chongqing 400038， China ().xjzslw@hotmail.com Drs Luo， Hou， Zhang， Xie， Wu， and Xu are from the Department of Orthopaedics， South-West Hospital， The Third Military University， Chongqing， China. Drs Luo， Hou， Zhang， Xie， Wu， and Xu have no relevant financial relationships to disclose. This study was supported by the National High Technology Research and Development Program (863 Project， Grant No.： 2006AA02A122) and the Chongqing Natural Sciences Foundation (Grant No.： 2010JJ0379). Correspondence should be addressed to： Jianzhong Xu， PhD， Department of Orthopaedics， South-West Hospital， The Third Military University， No. 29 Gaotanyan Rd， Chongqing 400038， China (xjzslw@hotmail.com). Bone tissue engineering involves the use of tissue engineering methods to promote the generation and differentiation of bony cells. Using this approach， new approaches can be explored to repair long segmental bone defects. Mesenchymal stem cells are one of the most widely used types of stem cells for bone tissue engineering. Following in vitro amplification using chemical induction to induce mesenchymal stem cell osteoblast differentiation， the resulting osteoblasts and support material can be combined to generate engineered bone tissue. In 1977， Bassett et al 1 developed pulsed electromagnetic field therapy and successfully treated a group of patients with bone nonunion. Since then， several studies have reported that pulsed electromagnetic fields accelerate the speed of mesenchymal stem cell amplification and osteoblast induction， growth factor secretion， and extracellular matrix synthesis; pulsed electromagnetic fields may also promote bone reconstruction and accelerate bone growth. 2–7 These findings suggest that further studies of the effects of pulsed electromagnetic fields on mesenchymal stem cell osteoblast differentiation and bone tissue engineering are needed. 8–13 These previous studies were performed using a specific pulsed electromagnetic field frequency during the course of the experiments; notably， the frequency is an important indicator for determining the biological effects of pulsed electromagnetic fields. In the current study， we investigated the effects of different pulsed electromagnetic field frequencies on mesenchymal stem cell induction in vitro with the overall goal of providing a new approach for mesenchymal stem cell induction in vitro and experimental support for a new type of bioreactor and the clinical application of pulsed electromagnetic fields to promote bone fracture healing. The results of the current study should provide a strong theoretical foundation for future pulsed electromagnetic field-related research. Materials and Methods The following solutions， kits， and equipment were used in the experiments described herein： Percoll solution (Sigma-Aldrich， St Louis， Missouri)， HyClone Dulbecco’s Modified Eagle Medium： Nutrient Mixture F-12 (DMEM/F12) (1：1) (Thermo Fisher Scientific Inc， Waltham， Massachusetts)， HyClone fetal calf serum (Thermo Fisher Scientific Inc)， alkaline phosphatase test kit (Nanjing Jiancheng Bioengineering Institute， Nanjing， China)， osteocalcin radioimmunity kit (Beijing China Atomic Research Institute， Beijing， China)， mouse-anti-human osteocalcin antibody (Boshide， Wuhan， China)， ultraviolet/visible spectrophotometer (UV751GD; the Shanghai Equipment Confactory， Shanghai， China)， and controllable pulsed electromagnetic field activator (Logistical Engineering University of PLA， Chongqing， China). Separation and Cultivation of Human Mesenchymal Stem Cells Bony marrow (5–10 mL) from both sides of the iliac crest was collected from healthy volunteers in a sterilized environment using heparin as the anticoagulant. Percoll solution (1.073 g/mL) was used to isolate the bony marrow. A total of 5×105 cells/cm 2 were inoculated into a culture flask containing DMEM/F12 supplemented with 15% fetal calf serum， and the cells were incubated under saturated humidity. After 3 generations， the cells were digested and centrifuged to generate a cell suspension， which was divided into 6 experimental groups and 1 control group. Treatment of Human Mesenchymal Stem Cells With Different Pulsed Electromagnetic Field Frequencies The 6 experimental cell groups were treated with a Helmholtz coil with pulsed electromagnetic fields， which was a dual coil with a 10-cm space between the 2 coils. The Hall effect was used to measure the magnetic reactor to confirm that the field’s homogeneity and stabilization were acceptable. The coil was placed into the cell incubator， and the field was set to different pulsed electromagnetic field frequencies， including 5， 25， 50， 75， 100， and 150 Hz， each with a field intensity of 1.1 mT， for 30 minutes per day for 21 days. The control groups of cells were incubated under the same experimental conditions with no exposure to the pulsed electromagnetic fields. Morphological Observations Changes in cell morphology under the different growth conditions were observed using an inverted microscope. Photos of cell morphology were taken on days 1， 3， 5， and 7 after serial subcultivation. Ultramicrostructural Observations Following exposure to pulsed electromagnetic fields， when the cells covered 80% of the area of the culture bottle， they were digested， centrifuged， and washed twice with phosphate buffered saline. The cells were immobilized with 2.5% glutaraldehyde and subjected to gradient acetone anhydration， followed by embedding in epoxy resin. Extra thin sections were generated and stained with uranyl acetate and lead citrate. Transmission electron microscopy was used to observe the ultramicrostructure of the experimental and control groups. Alkaline Phosphoric Acid Enzyme Staining When using Gomori stain， cells positive for alkaline phosphatase expression are brownish-black. Following 6 days of pulsed electromagnetic field stimulation， the human mesenchymal stem cells were removed from the incubator， rinsed with phosphate buffered saline， fixed in ice-cold acetone (−20°C)， incubated for 3 hours at 37°C in phosphoric acid enzyme liquids， washed with distilled water， and subjected to ammonium sulfide processing using 2% nitric acid and 1% cobalt， 1 by 1 in order， followed by staining with neutral and red contrast dye. Alkaline Phosphatase Activity Measurements After 3， 6， 9， 12， and 15 days of pulsed electromagnetic field exposure， cells grown in 12-well flat-bottomed plates from each group were digested， centrifuged， collected， and washed twice with phosphate buffered saline. The cell density was adjusted to 1×105 cells/mL in 0.5% Triton-X100 (Sigma-Aldrich， St Louis， Missouri)， and the cells were incubated at 4°C for 12 hours. Intermittent ultrasound exposure on ice was used to break up the cells and ensure complete cell lysis (150 W， 5 s). The absorbance of 50 μL of the cell lysate was examined at 520 nm to determine the A value， and a standard formula was used to calculate alkaline phosphatase activity. Collagen Type I and Osteocalcin Immunostaining Coverslips were removed after 12 days， followed by phosphate buffered saline rinses， fixation with 95% ethanol， and treatment with 3% hydrogen dioxide， followed by rinses with distilled water and incubation with 5% normal goat serum. Cells were subjected to immunohistochemical staining using the streptavidin-biotin peroxidase complex method with a rat anti-collagen type I and anti-osteocalcin monoclonal antibody. Quantification of Osteocalcin Levels At 7， 14， and 21 days， 50 mL were removed from each culture， followed by digestion with diastase vera. Following centrifugation， a total of 1×106 cells were resuspended in 1 mL of a 1：1 admixture of phosphate buffered saline and Triton-X100， followed by incubation at 4°C overnight. The resulting samples were sent to the Atomic Medical Department of Southwest Hospital to measure the osteocalcin content in the cells using a radiographic-immunity method based on the radiographic-immunity competitive binding principle. In this assay， 125 I-marked osteocalcin competes with free osteocalcin for binding to the osteocalcin antibody， and a ϒ counter is used to measure the sediment counts per minute， which is used to determine the osteocalcin content of the sample according to a standard curve. Staining With Alizarin Monosulfonate Calcium Dye After the appearance of oval-shaped nodules in the 6-well culture plate， the coverslips were removed， rinsed with phosphate buffered saline， fixed with 75% alcohol for 30 minutes， stained with 2% alizarin monosulfonate， dehydrated using an alcohol gradient， made transparent with xylene， and mounted using neutral resin. Staining With Von Kossa Calcium Dye After oval-shaped nodules appeared in the 6-well culture plate， the coverslips were removed， rinsed with phosphate buffered saline， fixed with 75% alcohol， immersed in 2% silver nitrate aqua， exposed to ultraviolet light for 30 minutes， washed with distilled water， stained with neutral red dye， treated with an alcohol gradient， made transparent with dimethyl benzene， and sealed using a neutral resin. Staining With Achromycin Dye After oval-shaped nodules appeared in the 6-well culture plate， the coverslips were removed， treated with 0.1 mg/mL achromycin culture solution for 30 minutes， incubated with a common culture solution for 30 minutes， rinsed with phosphate buffered saline， fixed with 75% alcohol， and observed using a fluorescence microscope. Statistical Analyses SPSS version 11.0 software (IBM， Armonk， New York) was used for statistical analyses. Self-paired t analysis was used to analyze each set of experimental data. One-factor analysis of variance was used to analyze different groups stimulated with different pulsed electromagnetic fields， and F analysis was used to complete the statistical analysis. Results Inverted Phase Contrast Microscope Observations At 4 hours post-inoculation， the third-generation human mesenchymal stem cells were adhered to the plates， and they began to divide and grow 24 hours after they adhered. After 3 days， pulsed electromagnetic field group cells were larger than control group cells， and their morphology continued to change; the cells eventually became triangular and polygonal in shape， scales formed， and the cytoplasm contained abundant matrix and granular material. No obvious differences were observed in the appearances of pulsed electromagnetic field group cells compared with control group cells. Over time， the cells became confluent and began to exhibit overlapping growth. After 3 weeks， mineralization of the matrix led to the fusion of the oval-shaped calcified nodules. Around the nodules， the cells were distributed in an array-like pattern. Ultramicrostructural Observations Transmission electron microscopy analysis of human mesenchymal stem cells in the pulsed electromagnetic field group showed that they were more differentiated than the control group cells. The nuclear matrix ratio of pulsed electromagnetic field group cells was lower than that of control group cells. The cytoplasm of pulsed electromagnetic field group cells contained abundant organelles， including mitochondria， rough endoplasmic reticulum， and Golgi bodies. Control group human mesenchymal stem cells were more immature with larger nuclei， a similar nuclear-matrix ratio， and fewer organelles. Alkaline Phosphatase Staining Cells that were not stimulated with pulsed electromagnetic fields were negative for alkaline phosphatase expression， whereas cells subjected to pulsed electromagnetic field stimulation were highly positive for alkaline phosphatase expression， with brownish-black cytoplasm and black granulated precipitates. Alkaline Phosphatase Activity Measurements Pulsed electromagnetic field group cells exhibited stronger alkaline phosphatase activity than control group cells starting on the third day. As time progressed， alkaline phosphatase activity was higher in pulsed electromagnetic field group cells， reaching a peak at 12 days. By day 15， no great change had occurred， and alkaline phosphatase activity remained stable， although it remained significantly higher than that of the control groups (Figure 1 ). Furthermore， different frequencies were associated with different alkaline phosphatase activity levels. Alkaline phosphatase activity in the 50-Hz pulsed electromagnetic field group was higher than that of the other pulsed electromagnetic field groups on days 9， 12， and 15. Figure 1： Alkaline phosphatase (ALP) activity in human mesenchymal stem cells treated with different frequencies of pulsed electromagnetic fields (PEMF). Collagen Type I and Osteocalcin Immunohistochemical Staining After 12 days of pulsed electromagnetic field stimulation， human mesenchymal stem cells subjected to collagen type I immunohistochemical staining exhibited high levels of yellow granulation and were highly positive. In contrast， no yellow granulation was detected in the control group cells. Osteocalcin immunohistochemical staining of human mesenchymal stem cells showed that they exhibited high levels of yellow granulation and were highly positive. In contrast， no yellow granulation was detected in the control group cells. Quantification of Osteocalcin Levels On day 7， cells in all of the different pulsed electromagnetic field groups expressed